Hepatic Seipin depletion increases SCD1 activity and LD formation 1 Hepatic BSCL2 (Seipin) deficiency disrupts lipid droplet homeostasis and increases lipid metabolism via SCD1 activity Mohamed Amine Lounis 1 , Simon Lalonde 1 , Sabri Ahmed Rial 1 , Karl-F. Bergeron 1 , Jessica C. Ralston 2 , David M. Mutch 2 , and Catherine Mounier 1 1 BioMed Research Center, Biological Sciences Department, University of Quebec in Montreal (UQÀM), Montreal, Quebec, Canada 2 Department of Human Health & Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada Running title: Hepatic Seipin depletion increases SCD1 activity and LD formation Corresponding author: Prof. Catherine Mounier, Département des sciences biologiques et centre de recherche BioMed, Université du Québec à Montréal, Case Postale 8888, Succursale Centre-ville, Montréal, QC, H3C 3P8, Canada. Phone: 1 (514) 987-3000 extension 8912. Fax: 1 (514) 987-4647. E-mail: [email protected]Key words: BSCL2, Seipin, lipid droplets, SCD1, lipogenesis, fatty acid uptake, insulin sensitivity
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Hepatic Seipin depletion increases SCD1 activity and LD formation
1
Hepatic BSCL2 (Seipin) deficiency disrupts lipid droplet homeostasis and increases lipid metabolism via SCD1 activity
Mohamed Amine Lounis1, Simon Lalonde1, Sabri Ahmed Rial1, Karl-F. Bergeron1,
Jessica C. Ralston2, David M. Mutch2, and Catherine Mounier1
1 BioMed Research Center, Biological Sciences Department, University of Quebec in
Montreal (UQÀM), Montreal, Quebec, Canada
2 Department of Human Health & Nutritional Sciences, University of Guelph, Guelph,
Agilent Technologies). Fatty acid peaks were identified by comparison with retention
times of fatty acid methyl ester standards. To estimate SCD1 activity, we calculated the
product-to-precursor fatty acid ratio (i.e., 18:1n9/18:0 and 16:1n7/16:0), as previously
reported [46, 47]. Fatty acid data were normalized to protein concentrations for each
treatment condition and reported as µg fatty acid per µg/µl protein.
Fatty acid uptake
Uptake of [3H]oleate was measured in confluent HepG2 cells and rat primary hepatocytes
as previously described [48, 49]. Briefly, 0.68µCi of [9,10-3H]oleic acid (54.6 Ci/mmol;
Perkin Elmer) was mixed with 50µM of non-radioactive oleate (Sigma-Aldrich) and
dissolved in a 173µM BSA solution free of fatty acids. Cells were incubated with this
oleate/BSA solution for 10min at 37°C. Uptake was stopped by removal of the
oleate/BSA solution followed by the addition of ice-cold 1x PBS (5ml) containing
200µM of phloretin and 0.1% BSA (wt/v). After a 2min incubation, cells were washed
six times with ice-cold 1x PBS. Cells were then lysed with 2M NaOH and aliquots of the
lysate were used for protein concentration and radioactivity measurements. Radioactivity
was measured in a TRI Carb 2800TR liquid scintillation counter after the addition of
10ml Ultima-Gold (Perkin Elmer). Data were presented as number of counts per minute
Hepatic Seipin depletion increases SCD1 activity and LD formation
12
(CPM) of [3H] per µg of protein.
De novo lipogenesis
De novo lipogenesis was evaluated by measuring the incorporation of [14C]acetate into
lipids, as described previously [44, 50, 51]. Briefly, post-transfection cells were incubated
with 1µCi of [1,2-14C]acetic acid (54.3 Ci/mol; Perkin Elmer) for 4h. Cells were then
suspended in 200µl 1x PBS and total cellular lipids were extracted in
chloroform/methanol (2:1, v/v). The lipid extract was dried under nitrogen and
reconstituted in 100µl hexane. Radiolabeled lipids were separated by thin layer
chromatography on silica-coated plates using a hexane/diethyl-ether/acetic acid solution
(80:20:1, v/v) as a developing solvent [44, 50, 51]. Lipids were visualized by exposure to
iodine vapors and the bands corresponding to authentic lipid standards (FFA, TAG,
DAG, CE, and PL) were scraped into separate vials. Radioactivity was measured and data
presented as CPM of [14C] per µg of protein.
Statistical analysis
When evaluating statistical significance, we used a Student’s t-test (two-tailed) to
compare two groups and a two-way analysis of variance (ANOVA) when more than one
factor was evaluated. A p<0.05 was considered statistically significant. Unless specified
otherwise, data are presented as mean ± SD.
Hepatic Seipin depletion increases SCD1 activity and LD formation
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Results
Seipin is expressed in hepatocytes
Since Seipin as yet to be shown to play a cell autonomous role in the liver, our first aim
was to confirm the presence of the Seipin protein in the various hepatic models used in
this study. To this end, we performed antibody-based analyses in HepG2 cells (a human
hepatocyte-hepatocarcinoma cell line) as well as in rat primary hepatocytes. The testis, a
tissue known to express Seipin [52], was used as positive control. An anti-Seipin
antibody (Table 1), detected a band of ~67kD in the rat testis and a major band at ~60kD
in the rat adipose tissue, in agreement with previous observations [52]. Primary rat
hepatocytes also showed a ~60kD band while HepG2 cells displayed a slightly higher
band (~61kD), presumably reflecting the fact that human Seipin is 33 amino acids longer.
The detected levels of Seipin were lower in adipocytes and hepatocytes compared to
testis (Fig.1A, left panels). A ~60kD band detected in liver extracts of WT mice was
absent in liver extracts of Bscl2-/- mice (Fig.1A, right panels), confirming antibody
specificity. We used immunohistochemistry to expand and confirm these results. The
Seipin protein was concentrated in the perinuclear space of HepG2 cells, a pattern
consistent with ER localization (Fig.1B).
BSCL2 knockdown alters lipid droplet morphology and size
To evaluate the role of Seipin in vitro, primary rat hepatocytes and HepG2 cells were
transfected with a siRNA designed to knockdown Bscl2/BSCL2. This approach decreased
Seipin protein levels to 54% and 36% in HepG2 cells (Fig.1C, left panels) and primary
rat hepatocytes (Fig.1C, right panels), respectively. Oleate treatment was then used to
induce LD formation, increasing both number and size of the organelle (Fig.2A).
Inhibition of Bscl2/BSCL2 expression increased the number (80% more) and the size
(150% larger) of LD, irrespective of the presence of oleate, in both rat primary
hepatocytes and HepG2 cells (Fig.2A). Live imaging of Seipin-deficient HepG2 cells
showed greater LD clustering with at least partial LD fusion, resulting in aggregates of
Hepatic Seipin depletion increases SCD1 activity and LD formation
14
abnormal morphology, and suggested increased LD persistence (Fig.2B). We analyzed
mRNA expression levels of key genes implicated in LD homeostasis. In both HepG2
cells and the liver of Bscl2-/- mice (Fig.3), a Seipin deficiency was associated with
increased expression of Plin5/PLIN5 and Cidea/CIDEA; genes involved in LD formation
and stability, respectively [53, 54]. In contrast, a decrease in Atgl/ATGL expression, a
gene known to be involved in lipolysis [55-57], was observed in HepG2 cells and the
liver of fasted Bscl2-/- mice, but not in fed mice. Together, our data suggest that lowering
Seipin levels in hepatocytes increased LD biogenesis.
Alteration of lipid droplet homoeostasis following BSCL2 knockdown is concomitant
with changes in lipid metabolism
In order to better understand the mechanism underlying the observed increase in LD
formation and expansion in Seipin-deficient cells (Fig.2), we evaluated fatty acid uptake
by [3H]oleate incorporation into HepG2 cells and primary rat hepatocytes following
siRNA transfection. Bscl2/BSCL2 knockdown caused an increase in fatty acid uptake in
both HepG2 cells and primary rat hepatocytes (40%; Fig.4A). In siRNA-transfected
HepG2 cells, elevated fatty acid uptake was associated with increased levels of the
PPARγ transcription factor (40%; Fig.4B, top panel) and at least one of its direct targets,
the translocase CD36 implicated in fatty acid transport [58] (80%; Fig.4B, middle panel).
It has recently been shown that Seipin can have an effect on PPARγ nuclear localization
and activity through an interaction with the TAG synthesis enzyme AGPAT2 [30]. We
therefore investigated AGPAT2 levels in our siRNA-transfected cells. Interestingly,
Seipin deficiency led to increased expression of AGPAT2 (40%; Fig.4B, bottom panel).
To determine if de novo lipogenesis was also affected by diminished Seipin levels, we
measured [14C]acetate incorporation into lipids of siRNA-transfected hepatic cells. A
25% increase in the synthesis of total lipids was observed in HepG2 cells transfected with
BSCL2 siRNA (Fig.5A, top panel). This higher overall synthesis was primarily due to
increased synthesis of TAG and DAG (22% and 28% respectively; Fig.5A, bottom
panels). No difference was observed in the other lipid fractions examined, such as free
fatty acids, ceramides and cholesterol esters (data not shown). In siRNA-transfected
Hepatic Seipin depletion increases SCD1 activity and LD formation
15
HepG2 cells, elevated de novo lipogenesis was also associated with increased expression
of several lipogenic enzymes such as ACC, FAS and SCD1, as well as the transcription
factor SREBP-1c (Fig.5B). In addition, an increase in mature SREBP-1c protein levels
was observed in the liver of Bscl2-/- mice (Fig.5C). Interestingly, we also showed a
decrease in the expression of proteins implicated in lipolysis [55-57] and β-oxidation [59,
60] (ATGL and PPARα, respectively) (Fig.5B, bottom panels), suggesting that inhibition
of β-oxidation may contribute to the LD alterations observed in Seipin-deficient cells.
Seipin and SCD1 have opposing effect on LD formation and lipid synthesis
We showed an induction of SCD1 expression in Seipin-deficient cultured hepatocytes
(Fig.5B). A similar result was reported in the liver of Bscl2-/- mice [8]. The mostly
perinuclear pattern of expression observed for both SCD1 and Seipin is consistent with
the expected localization of these two ER-resident proteins. We determined that SCD1
and Seipin colocalize in HepG2 cells (Pearson’s r value: 0.63, Spearman's p value: 0.47;
Fig.6A), hinting to a possible functional relationship between these two proteins. We
therefore tested the effect of SCD1 deficiency in HepG2 cells using a validated siRNA
(Fig.6B). Co-transfection of SCD1 and BSCL2 siRNA rescued the Seipin deficiency
phenotype, i.e., the number and size of LD was close to normal when compared to cells
transfected with BSCL2 siRNA alone, irrespective of the presence or absence of oleate
(Fig.6C). The increase in fatty acid uptake (Fig.6D) and de novo lipogenesis (Fig.6E)
observed in Seipin-deficient HepG2 cells was also lost when cells were co-transfected
with SCD1 and BSCL2 siRNA. Co-transfected cells exhibited a low lipid uptake and
synthesis profile closer to that of cells transfected with SCD1 siRNA alone. In a similar
fashion, we found that the MUFA/SFA ratio is higher in Seipin-deficient hepatocytes
compared to non-transfected control HepG2 cells, whereas it tended to be lower in
SCD1-deficient or co-transfected cells (Fig.6F). Together, these observations show that
Seipin and SCD1 have opposing effects on LD formation and lipid synthesis, and suggest
that Seipin’s effect on hepatic lipid synthesis/accumulation is mediated by SCD1.
Seipin deficiency increases basal phosphorylation of insulin-signaling proteins
Hepatic Seipin depletion increases SCD1 activity and LD formation
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General insulin resistance and hyperglycemia are characteristic of patients with BSCL2
[5, 17, 61], as well as Bscl2-/- mice [7-9]. We therefore evaluated the expression level and
the phosphorylation state of key proteins involved in the insulin-signaling cascade. Seipin
deficiency was associated with increased phosphorylation of AKT (both Ser473 and
Thr308 sites), ERK1/2, mTOR and P70S6K in HepG2 cells stimulated or not by insulin
(Fig.7A). The levels of AKT (Ser473) and IRS1 (Tyr612) phosphorylation were also
increased in liver extracts of Bscl2-/- mice (Fig.7B). In accordance with an increase in the
phosphorylation of insulin-signaling proteins, we noted that, in Bscl2-/- mice, the
expression of glucose metabolism markers such as Gck was increased while G6pc
expression was decreased. Unexpectedly, Pepck expression was increased (Fig.7C).
We then analyzed the effect of BSCL2 knockdown on physiological responses activated
by insulin. We stimulated siRNA-transfected rat primary hepatocytes with insulin and
measured both fatty acid uptake and de novo lipogenesis. In accordance with initial
observations (Figs.4&5), Seipin deficiency increased both fatty acid uptake and
lipogenesis. However, the presence of insulin further stimulated fatty acid uptake (25%
compared to BSCL2 siRNA-transfected cells without insulin) (Fig.7D) and de novo
lipogenesis (35% total lipids, compared to BSCL2 siRNA-transfected cells without
insulin) (Fig.7E). As previously observed (Fig.5A), increased de novo lipogenesis
primarily stemmed from increased TAG and DAG synthesis (Fig.7E), as the levels of
others lipid classes did not vary (data not shown). These data suggest that in cases of
Seipin deficiency, hepatic lipid uptake and synthesis as well as gluconeogenesis are
increased, probably aggravating the BSCL2 phenotype and contributing to hepatic
dysfunction.
Seipin deficiency increases the expression of ER stress markers
ER stress and the unfolded protein response (UPR) are critically involved in the initiation
of many diseases, such as the metabolic syndrome [40, 62, 63]. In addition, this pathway
has been reported to play an important role in LD formation and lipogenesis promotion in
the liver [39, 64]. To determine if Seipin deficiency could induce ER stress, we measured
the mRNA levels of key genes implicated in this process. In BSCL2 siRNA-transfected
Hepatic Seipin depletion increases SCD1 activity and LD formation
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HepG2 cells, ATF4 and GRP78 mRNA expression was increased by a little over 50% and
CHOP by 100% (Fig.8A). PERK protein levels were also increased by 34% and 27% in
BSCL2/Bscl2 siRNA-transfected HepG2 cells as well as primary rat hepatocytes,
respectively (Fig.8B). Our study revealed that Seipin deficiency induces expression of
several markers of ER stress.
Hepatic Seipin depletion increases SCD1 activity and LD formation
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Discussion
In this study, we first confirmed that Bscl2 is expressed in mouse liver (Fig.1A) albeit at
a low protein level relative to testis, as previously reported for mRNA [65]. One group
was recently able to detect the Seipin protein in human liver using LC/MS
(humanproteomemap.org; [66]). We used siRNA to lower Seipin protein levels in
hepatocytes (Fig.1C). This induced an increase in the number and the size of LD
(Fig.2A). We also observed LD aggregation defects (Fig.2B), implying that Seipin plays
a role in the generation, expansion and morphology of LD. These observed changes in
shape and number of LD are similar to reported results in yeast, where a depletion of the
BSCL2 yeast ortholog SEI1/FLD1 led to LD with irregular shapes and sizes [19, 21, 22,
24]. Up to 30% of the SEI1/FLD1 deletion mutants contained one or a few supersized LD
and about 60% of them contained an amorphous aggregation of LD [19].
Seipin has been suggested to act as a scaffold protein [30], however its function remains
unclear. Seipin and SCD1 proteins are both localized in the ER [67, 68], and colocalize
significantly in HepG2 cells (Fig.6A). Moreover, Seipin and SCD1 have opposite effects
on LD homeostasis (Fig.6C). Taken together, our results suggest that Seipin and SCD1
are part of an ER-resident protein complex that controls LD formation. Different models
for LD biogenesis consistently suggest that LD emerge from the ER [69]. The most
accepted model posits that the accumulation of TAG between the bilayer leaflets of the
ER membrane drives the genesis of nascent LD [28, 29]. In accordance with this, LD
have been localized in close proximity to or even tethered to the ER in yeast [70, 71].
Seipin deficiency in hepatocytes caused lipid accumulation and was associated with
increased expression of CHOP, GRP78, ATF4 and the protein PERK (Fig.8), four
markers of the unfolded protein response (UPR) to ER stress. A similar consequence of
Seipin deficiency was previously observed in neuronal cells [72, 73]. In these prior
studies, loss-of-function mutations in the Seipin protein induced a “seipinopathy”, a
motor neuron disease associated with high LD formation and TAG storage as well as ER
stress [74]. Interestingly, an activation of the UPR (PERK-eIF2α-ATF4) pathway, like
the one seen in our in vitro models (Fig.8), can activate the expression of several
Hepatic Seipin depletion increases SCD1 activity and LD formation
19
lipogenic genes (Acc/ACC, Fas/FAS, Scd1/SCD1) and the associated transcription factor
SREBP-1c [11, 33, 35-37], thereby accentuating hepatic lipid synthesis/storage and
potentially aggravating a nascent hepatic steatosis [33, 35, 37, 38].
The presence of smaller than normal LD in C.elegans fat-6;fat-7 double mutants lacking
most desaturases [75] suggests SCD activity is required for LD expansion. The
MUFA/SFA intracellular lipid ratio also seems to play an important role in LD
homeostasis, most notably on fusion and growth [76]. This is consistent with a role for
SCD1, an enzyme that converts SFA into MUFA, in LD biogenesis and, by extension,
TAG storage. For example, an increase in LD size within 3T3-L1 preadipocyte cells is
associated with an increase in SCD1 expression and MUFA/SFA ratio [76]. The increase
in SCD1 expression (Fig.5B) following our BSCL2 knockdown was concomitant with an
increase in the MUFA/SFA ratio (hence SCD1 activity; Fig.6F) and TAG synthesis
(Figs.5A and 7E).
Moreover, high Scd1 expression activates de novo lipogenesis via an elevation in hepatic
SREBP-1c levels [77] that consequently increases the expression of lipogenic genes,
including Scd1 itself. The potential therefore exists for a reinforcing feedback loop to be
established, leading to Scd1 overexpression and enhancing hepatic lipogenesis following
Seipin deficiency (Fig.5). Underlying the important role of SCD1 in TAG metabolism,
DGAT (key enzymes implicated in TAG synthesis) colocalize with SCD1 in the ER [78]
and could have synergistic interactions with Seipin [26]. The increase in number and size
of LD we observed following BSCL2 knockdown (Figs.2A&6C) could be due to lipid
metabolism changes secondary to an increase in SCD1 activity. In agreement with this
hypothesis, 3T3-L1 preadipocyte cells bearing a Bscl2 mutation, and presumably
possessing increased SCD1 activity, exhibit enhanced TAG synthesis [25]. Overall,
increased SCD1 activity appears sufficient to explain most of the observed effects on
fatty acid metabolism following Bscl2/BSCL2 knockdown, including elevated PPARγ
expression and CD36-mediated fatty acid uptake (Fig.4). In accordance with this, a SCD1
knockdown reversed the effect of Seipin deficiency on LD formation (Fig.6C) and on
lipid metabolism (Fig.6D,E) in hepatocytes. Inhibition of SCD1 activity is known to
decrease PPARγ expression [79]. As CD36, the key cell surface receptor that facilitates
Hepatic Seipin depletion increases SCD1 activity and LD formation
20
hepatic fatty acid uptake, is a direct transcriptional target of PPARγ [58], it is not
surprising that SCD1 deficiency alone decreases oleate uptake (Fig.6D).
Recent studies have shown that the adipose tissue plays a major role in the establishment
of hepatic steatosis in Bscl2-/- mice [7, 9, 15]. Expressing Seipin in adipose tissue alone is
sufficient to rescue lipodystrophy, hepatic steatosis and insulin resistance in Bscl2-/- mice
[15]. Chen and collaborators also reported that mice with a specific hepatic deletion of
Seipin (Bscl2Li-/-) did not show increased lipid deposition in the liver on a standard chow
diet [80]. Such striking results led these researchers to conclude that Seipin does not play
a role in hepatic steatosis. However, our study clearly shows an effect of Seipin
deficiency on fat accumulation in hepatocytes. The discrepancy between these in vivo
results and our observations is probably due to the storage of circulating lipids, in the
form of free or esterified fatty acids in the blood, within the adipose tissue. In Bscl2-/-
mice, the absence of adipose tissue causes an accumulation of plasma fatty acids and a
compensatory fatty acid uptake (leading to steatosis) in the liver. Consequently, Bscl2-/-
mice do not suffer from hypertriglyceridemia [7, 8]. Bscl2Li-/- mice possess a normal
adipose tissue that stores excess fat originating from a standard chow diet. However, a
high fat diet leads to a strong increase in circulating lipid levels, and under these
conditions both Bscl2Li+/+ and Bscl2Li-/- mice develop hepatic steatosis [80]. Presumably,
the storage capacity of both the adipose tissue and the liver becomes saturated in these
mice, precluding the observation of more subtle intracellular differences. Consistent with
a cell autonomous role for Seipin in hepatic fat accumulation, we show that the
expression of several genes implicated in LD homeostasis is elevated in the liver of
Bscl2-/- mice (Plin5 and Cidea; Fig.3). Seipin does not appear to be necessary for the
formation of a LD, notably in Bscl2/BLSC2-/- hepatocytes, but this does not exclude a role
in lipid storage. In support of such a role, Yang et al. have suggested that Seipin restricts
lipogenesis and LD accumulation in non-adipocyte cells [27]. The authors show that
Seipin overexpression inhibits ectopic lipid-induced LD formation in a mouse hepatocyte
cell line (AML-12 cells) and distinguish two functions for the Seipin protein, one in
adipocyte maturation (via its C-terminal domain) and another in the control of
intracellular lipid levels (via a conserved core sequence) in non-adipocyte cells [27].
Therefore, we argue that the endogenously expressed Seipin protein plays a role in LD
Hepatic Seipin depletion increases SCD1 activity and LD formation
21
homeostasis and TAG storage in hepatocytes.
Several previous studies have clearly demonstrated that Seipin deficiency in both mice
and humans leads to insulin resistance [7-9]. Surprisingly, we observed that Seipin
deficiency increased basal phosphorylation of AKT, ERK and P70S6K proteins,
suggesting an increase in insulin sensitivity (Fig.7A). In agreement with this
interpretation, Chen and collaborators observed improved hepatic insulin signaling in
Bscl2-/- mice, as measured by insulin clamp [16]. To explain their result, the authors
suggest that the enhanced insulin sensitivity observed after 16h fasting stems from
increased levels of insulin receptors and downstream signaling effectors such as IRS1 and
AKT [16]. The increased basal phosphorylation of insulin signaling pathway proteins
observed in our BSCL2 siRNA-transfected hepatocytes may be explained, at least in part,
by the effect of Seipin deficiency on SCD1 activity (Fig.6F), as an increase in MUFA has
been shown to stimulate insulin signaling [81-85].
Insulin negatively regulates the expression of gluconeogenic genes Pepck and G6pc, and
increases expression of Gck, the enzyme responsible for the first step of hepatic
glycolysis [86]. The modulation of glucose metabolism markers observed in our study
(Fig.7C) is somewhat consistent with an activation of the insulin signaling proteins
following Seipin deficiency, with the notable exception of Pepck gene expression being
specifically elevated in fasted mice. This paradox might be explained by the activation of
ER stress in Seipin deficient hepatocytes (Fig.8). PEPCK expression is activated by ER
stress through promoter binding by the ATF4 transcription factor, leading to increased
transcription [87]. Additionally, Pepck expression could enhance gluconeogenesis in
Seipin deficient cells, leading to increased intracellular glucose concentration and the
reduction of AMPK phosphorylation we observe (Fig.7B) [88, 89]. Interestingly,
decreased AMPK phosphorylation/activity can also lead to an activation of its targets
proteins, notably the lipogenic enzyme ACC [90]. Therefore, a combined increase in
gluconeogenesis and insulin signaling can lead to further activation of lipogenic markers
such as FAS, SCD1 and ACC (Fig.5) [91-97].
We showed that Seipin depletion in cultured hepatocytes leads to an increase in LD
number and size. These changes in LD homeostasis are probably due to an upregulation
Hepatic Seipin depletion increases SCD1 activity and LD formation
22
of lipid metabolism, characterized by an increase in SCD1 activity leading to a higher
MUFA/SFA ratio. In accordance with this, a SCD1 knockdown reversed the LD
formation defects and the changes in lipid metabolism homeostasis associated with
Seipin depletion. Interestingly, Seipin and SCD1 also colocalize, leading us to propose a
functional interaction within the ER membrane whereby Seipin controls lipid metabolism
and storage through SCD1 activity and LD formation.
Hepatic Seipin depletion increases SCD1 activity and LD formation
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Acknowledgements
We wish to thank Dr. Jocelyne Magre (University of Nantes, France) for kindly
providing us with mRNA and protein extracts from Bscl2-/- mice, as well as Dr. Xiaoqin
Ye (University of Georgia, USA) for samples of Bscl2-/- mouse liver. We also thank
Denis Flipo for his precious help with confocal microscopy, the laboratory of Dr. Diana
Averill for primary rat hepatocytes and Dr. Daniel Boismenu for his help with data
analysis and discussion.
The Discovery Grants Program of the National Science and Engineering Research
Council of Canada (NSERC) funded this research. MAL and SL were supported by the
Fond de Recherche du Québec-Nature et Technologie (FRQNT) fellowships.
Conflict of Interest
The authors declare that they have no competing interests.
Hepatic Seipin depletion increases SCD1 activity and LD formation
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References
1. Garg, A., Clinical review#: Lipodystrophies: genetic and acquired body fat disorders. J Clin Endocrinol Metab, 2011. 96(11): p. 3313-25.
2. Berardinelli, W., An undiagnosed endocrinometabolic syndrome: report of 2 cases. J Clin Endocrinol Metab, 1954. 14(2): p. 193-204.
3. Seip, M., Lipodystrophy and gigantism with associated endocrine manifestations. A new diencephalic syndrome? Acta Paediatr, 1959. 48: p. 555-74.
4. Agarwal, A.K. and A. Garg, Seipin: a mysterious protein. Trends Mol Med, 2004. 10(9): p. 440-4.
5. Magre, J., et al., Identification of the gene altered in Berardinelli-Seip congenital lipodystrophy on chromosome 11q13. Nat Genet, 2001. 28(4): p. 365-70.
6. Agarwal, A.K. and A. Garg, Congenital generalized lipodystrophy: significance of triglyceride biosynthetic pathways. Trends Endocrinol Metab, 2003. 14(5): p. 214-21.
7. Cui, X., et al., Seipin ablation in mice results in severe generalized lipodystrophy. Hum Mol Genet, 2011. 20(15): p. 3022-30.
8. Prieur, X., et al., Thiazolidinediones partially reverse the metabolic disturbances observed in Bscl2/seipin-deficient mice. Diabetologia, 2013. 56(8): p. 1813-25.
9. Chen, W., et al., Berardinelli-seip congenital lipodystrophy 2/seipin is a cell-autonomous regulator of lipolysis essential for adipocyte differentiation. Mol Cell Biol, 2012. 32(6): p. 1099-111.
10. Payne, V.A., et al., The human lipodystrophy gene BSCL2/seipin may be essential for normal adipocyte differentiation. Diabetes, 2008. 57(8): p. 2055-60.
11. Liu, L., et al., Adipose-specific knockout of SEIPIN/BSCL2 results in progressive lipodystrophy. Diabetes, 2014. 63(7): p. 2320-31.
12. Unger, R.H., Lipotoxic diseases. Annu Rev Med, 2002. 53: p. 319-36. 13. van Herpen, N.A. and V.B. Schrauwen-Hinderling, Lipid accumulation in non-
adipose tissue and lipotoxicity. Physiol Behav, 2008. 94(2): p. 231-41. 14. Szendroedi, J. and M. Roden, Ectopic lipids and organ function. Curr Opin
Lipidol, 2009. 20(1): p. 50-6. 15. Gao, M., et al., Expression of seipin in adipose tissue rescues lipodystrophy,
hepatic steatosis and insulin resistance in seipin null mice. Biochem Biophys Res Commun, 2015. 460(2): p. 143-50.
16. Chen, W., et al., Molecular mechanisms underlying fasting modulated liver insulin sensitivity and metabolism in male lipodystrophic Bscl2/Seipin-deficient mice. Endocrinology, 2014. 155(11): p. 4215-25.
17. Cartwright, B.R. and J.M. Goodman, Seipin: from human disease to molecular mechanism. J Lipid Res, 2012. 53(6): p. 1042-55.
18. Fei, W., X. Du, and H. Yang, Seipin, adipogenesis and lipid droplets. Trends Endocrinol Metab, 2011. 22(6): p. 204-10.
19. Fei, W., et al., Fld1p, a functional homologue of human seipin, regulates the size of lipid droplets in yeast. J Cell Biol, 2008. 180(3): p. 473-82.
20. Wolinski, H., et al., A role for seipin in lipid droplet dynamics and inheritance in yeast. J Cell Sci, 2011. 124(Pt 22): p. 3894-904.
Hepatic Seipin depletion increases SCD1 activity and LD formation
25
21. Wolinski, H., et al., Seipin is involved in the regulation of phosphatidic acid metabolism at a subdomain of the nuclear envelope in yeast. Biochim Biophys Acta, 2015. 1851(11): p. 1450-64.
22. Wang, C.W., Y.H. Miao, and Y.S. Chang, Control of lipid droplet size in budding yeast requires the collaboration between Fld1 and Ldb16. J Cell Sci, 2014. 127(Pt 6): p. 1214-28.
23. Fei, W., et al., A role for phosphatidic acid in the formation of "supersized" lipid droplets. PLoS Genet, 2011. 7(7): p. e1002201.
24. Szymanski, K.M., et al., The lipodystrophy protein seipin is found at endoplasmic reticulum lipid droplet junctions and is important for droplet morphology. Proc Natl Acad Sci U S A, 2007. 104(52): p. 20890-5.
25. Fei, W., et al., Molecular characterization of seipin and its mutants: implications for seipin in triacylglycerol synthesis. J Lipid Res, 2011. 52(12): p. 2136-47.
26. Tian, Y., et al., Tissue-autonomous function of Drosophila seipin in preventing ectopic lipid droplet formation. PLoS Genet, 2011. 7(4): p. e1001364.
27. Yang, W., et al., Seipin differentially regulates lipogenesis and adipogenesis through a conserved core sequence and an evolutionarily acquired C-terminus. Biochem J, 2013. 452(1): p. 37-44.
28. Harris, C.A., et al., DGAT enzymes are required for triacylglycerol synthesis and lipid droplets in adipocytes. J Lipid Res, 2011. 52(4): p. 657-67.
29. Brasaemle, D.L. and N.E. Wolins, Packaging of fat: an evolving model of lipid droplet assembly and expansion. J Biol Chem, 2012. 287(4): p. 2273-9.
30. Talukder, M.M., et al., Seipin oligomers can interact directly with AGPAT2 and lipin 1, physically scaffolding critical regulators of adipogenesis. Mol Metab, 2015. 4(3): p. 199-209.
31. Yang, W., et al., BSCL2/seipin regulates adipogenesis through actin cytoskeleton remodelling. Hum Mol Genet, 2014. 23(2): p. 502-13.
32. Bi, J., et al., Seipin promotes adipose tissue fat storage through the ER Ca(2)(+)-ATPase SERCA. Cell Metab, 2014. 19(5): p. 861-71.
33. Puri, P., et al., Activation and dysregulation of the unfolded protein response in nonalcoholic fatty liver disease. Gastroenterology, 2008. 134(2): p. 568-76.
34. Puri, V., et al., Fat-specific protein 27, a novel lipid droplet protein that enhances triglyceride storage. J Biol Chem, 2007. 282(47): p. 34213-8.
35. Fang, D.L., et al., Endoplasmic reticulum stress leads to lipid accumulation through upregulation of SREBP-1c in normal hepatic and hepatoma cells. Mol Cell Biochem, 2013. 381(1-2): p. 127-37.
36. Rinella, M.E., et al., Dysregulation of the unfolded protein response in db/db mice with diet-induced steatohepatitis. Hepatology, 2011. 54(5): p. 1600-9.
37. Gentile, C.L., M. Frye, and M.J. Pagliassotti, Endoplasmic reticulum stress and the unfolded protein response in nonalcoholic fatty liver disease. Antioxid Redox Signal, 2011. 15(2): p. 505-21.
38. Zhang, X.Q., et al., Role of endoplasmic reticulum stress in the pathogenesis of nonalcoholic fatty liver disease. World J Gastroenterol, 2014. 20(7): p. 1768-76.
39. Zhang, K., et al., The unfolded protein response transducer IRE1alpha prevents ER stress-induced hepatic steatosis. EMBO J, 2011. 30(7): p. 1357-75.
Hepatic Seipin depletion increases SCD1 activity and LD formation
26
40. Zhang, C., et al., Endoplasmic reticulum-tethered transcription factor cAMP responsive element-binding protein, hepatocyte specific, regulates hepatic lipogenesis, fatty acid oxidation, and lipolysis upon metabolic stress in mice. Hepatology, 2012. 55(4): p. 1070-82.
41. Rutkowski, D.T. and R.J. Kaufman, A trip to the ER: coping with stress. Trends Cell Biol, 2004. 14(1): p. 20-8.
42. Harbrecht, B.G., et al., cAMP inhibits inducible nitric oxide synthase expression and NF-kappaB-binding activity in cultured rat hepatocytes. J Surg Res, 2001. 99(2): p. 258-64.
43. Harbrecht, B.G., et al., Glucagon inhibits hepatocyte nitric oxide synthesis. Arch Surg, 1996. 131(12): p. 1266-72.
44. Bligh, E.G. and W.J. Dyer, A rapid method of total lipid extraction and purification. Can J Biochem Physiol, 1959. 37(8): p. 911-7.
45. Ralston, J.C., et al., Inhibition of stearoyl-CoA desaturase-1 in differentiating 3T3-L1 preadipocytes upregulates elongase 6 and downregulates genes affecting triacylglycerol synthesis. Int J Obes (Lond), 2014. 38(11): p. 1449-56.
46. Fernandez, C., et al., Altered desaturation and elongation of fatty acids in hormone-sensitive lipase null mice. PLoS One, 2011. 6(6): p. e21603.
47. Attie, A.D., et al., Relationship between stearoyl-CoA desaturase activity and plasma triglycerides in human and mouse hypertriglyceridemia. J Lipid Res, 2002. 43(11): p. 1899-907.
48. Stremmel, W. and P.D. Berk, Hepatocellular influx of [14C]oleate reflects membrane transport rather than intracellular metabolism or binding. Proc Natl Acad Sci U S A, 1986. 83(10): p. 3086-90.
49. Pohl, J., A. Ring, and W. Stremmel, Uptake of long-chain fatty acids in HepG2 cells involves caveolae: analysis of a novel pathway. J Lipid Res, 2002. 43(9): p. 1390-9.
50. Bolker, H.I., et al., The incorporation of acetate-1-C14 into cholesterol and fatty acids by surviving tissues of normal and scorbutic guinea pigs. J Exp Med, 1956. 103(2): p. 199-205.
51. Jin, F.Y., V.S. Kamanna, and M.L. Kashyap, Niacin accelerates intracellular ApoB degradation by inhibiting triacylglycerol synthesis in human hepatoblastoma (HepG2) cells. Arterioscler Thromb Vasc Biol, 1999. 19(4): p. 1051-9.
52. Jiang, M., et al., Lack of testicular seipin causes teratozoospermia syndrome in men. Proc Natl Acad Sci U S A, 2014. 111(19): p. 7054-9.
53. Wu, L., et al., Cidea controls lipid droplet fusion and lipid storage in brown and white adipose tissue. Sci China Life Sci, 2014. 57(1): p. 107-16.
54. Wang, H., et al., Perilipin 5, a lipid droplet-associated protein, provides physical and metabolic linkage to mitochondria. J Lipid Res, 2011. 52(12): p. 2159-68.
55. Beller, M., et al., COPI complex is a regulator of lipid homeostasis. PLoS Biol, 2008. 6(11): p. e292.
56. Guo, Y., et al., Functional genomic screen reveals genes involved in lipid-droplet formation and utilization. Nature, 2008. 453(7195): p. 657-61.
57. Soni, K.G., et al., Coatomer-dependent protein delivery to lipid droplets. J Cell Sci, 2009. 122(Pt 11): p. 1834-41.
Hepatic Seipin depletion increases SCD1 activity and LD formation
27
58. He, J., et al., The emerging roles of fatty acid translocase/CD36 and the aryl hydrocarbon receptor in fatty liver disease. Exp Biol Med (Maywood), 2011. 236(10): p. 1116-21.
59. Cheon, Y., et al., Induction of overlapping genes by fasting and a peroxisome proliferator in pigs: evidence of functional PPARalpha in nonproliferating species. Am J Physiol Regul Integr Comp Physiol, 2005. 288(6): p. R1525-35.
60. Louet, J.F., et al., Long-chain fatty acids regulate liver carnitine palmitoyltransferase I gene (L-CPT I) expression through a peroxisome-proliferator-activated receptor alpha (PPARalpha)-independent pathway. Biochem J, 2001. 354(Pt 1): p. 189-97.
61. Berardinelli, S.D., R.M. Fischer, and I. Katz, Congenital absence of the pectoral muscle. Am J Roentgenol Radium Ther Nucl Med, 1956. 76(3): p. 599-604.
62. Thomas, S.E., et al., Diabetes as a disease of endoplasmic reticulum stress. Diabetes Metab Res Rev, 2010. 26(8): p. 611-21.
63. Kaufman, R.J., Orchestrating the unfolded protein response in health and disease. J Clin Invest, 2002. 110(10): p. 1389-98.
64. Lee, J.S., et al., Pharmacologic ER stress induces non-alcoholic steatohepatitis in an animal model. Toxicol Lett, 2012. 211(1): p. 29-38.
65. Chen, W., et al., The human lipodystrophy gene product Berardinelli-Seip congenital lipodystrophy 2/seipin plays a key role in adipocyte differentiation. Endocrinology, 2009. 150(10): p. 4552-61.
66. Kim, M.S., et al., A draft map of the human proteome. Nature, 2014. 509(7502): p. 575-81.
67. Lundin, C., et al., Membrane topology of the human seipin protein. FEBS Lett, 2006. 580(9): p. 2281-4.
68. Ntambi, J.M., et al., Loss of stearoyl-CoA desaturase-1 function protects mice against adiposity. Proc Natl Acad Sci U S A, 2002. 99(17): p. 11482-6.
69. Walther, T.C. and R.V. Farese, Jr., The life of lipid droplets. Biochim Biophys Acta, 2009. 1791(6): p. 459-66.
70. Novikoff, A.B., et al., Organelle relationships in cultured 3T3-L1 preadipocytes. J Cell Biol, 1980. 87(1): p. 180-96.
71. Perktold, A., et al., Organelle association visualized by three-dimensional ultrastructural imaging of the yeast cell. FEMS Yeast Res, 2007. 7(4): p. 629-38.
72. Ito, D. and N. Suzuki, Seipinopathy: a novel endoplasmic reticulum stress-associated disease. Brain, 2009. 132(Pt 1): p. 8-15.
73. Yagi, T., et al., N88S seipin mutant transgenic mice develop features of seipinopathy/BSCL2-related motor neuron disease via endoplasmic reticulum stress. Hum Mol Genet, 2011. 20(19): p. 3831-40.
74. Holtta-Vuori, M., et al., Alleviation of seipinopathy-related ER stress by triglyceride storage. Hum Mol Genet, 2013. 22(6): p. 1157-66.
75. Shi, X., et al., Regulation of lipid droplet size and phospholipid composition by stearoyl-CoA desaturase. J Lipid Res, 2013. 54(9): p. 2504-14.
76. Arisawa, K., et al., Changes in the phospholipid fatty acid composition of the lipid droplet during the differentiation of 3T3-L1 adipocytes. J Biochem, 2013. 154(3): p. 281-9.
Hepatic Seipin depletion increases SCD1 activity and LD formation
28
77. Miyazaki, M., et al., Hepatic stearoyl-CoA desaturase-1 deficiency protects mice from carbohydrate-induced adiposity and hepatic steatosis. Cell Metab, 2007. 6(6): p. 484-96.
78. Man, W.C., et al., Colocalization of SCD1 and DGAT2: implying preference for endogenous monounsaturated fatty acids in triglyceride synthesis. J Lipid Res, 2006. 47(9): p. 1928-39.
79. Kim, E., et al., Inhibition of stearoyl-CoA desaturase1 activates AMPK and exhibits beneficial lipid metabolic effects in vitro. Eur J Pharmacol, 2011. 672(1-3): p. 38-44.
80. Chen, W., et al., Molecular mechanisms underlying fasting modulated liver insulin sensitivity and metabolism in male lipodystrophic Bscl2/Seipin-deficient mice. Endocrinology, 2014: p. en20141292.
81. Coll, T., et al., Oleate reverses palmitate-induced insulin resistance and inflammation in skeletal muscle cells. J Biol Chem, 2008. 283(17): p. 11107-16.
82. Nardi, F., et al., Enhanced insulin sensitivity associated with provision of mono and polyunsaturated fatty acids in skeletal muscle cells involves counter modulation of PP2A. PLoS One, 2014. 9(3): p. e92255.
83. Xiao, C., et al., Differential effects of monounsaturated, polyunsaturated and saturated fat ingestion on glucose-stimulated insulin secretion, sensitivity and clearance in overweight and obese, non-diabetic humans. Diabetologia, 2006. 49(6): p. 1371-9.
84. Vessby, B., et al., Substituting dietary saturated for monounsaturated fat impairs insulin sensitivity in healthy men and women: The KANWU Study. Diabetologia, 2001. 44(3): p. 312-9.
85. Salvado, L., et al., Oleate prevents saturated-fatty-acid-induced ER stress, inflammation and insulin resistance in skeletal muscle cells through an AMPK-dependent mechanism. Diabetologia, 2013. 56(6): p. 1372-82.
86. O'Brien, R.M. and D.K. Granner, Regulation of gene expression by insulin. Physiol Rev, 1996. 76(4): p. 1109-61.
87. Mendez-Lucas, A., et al., Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) is a pro-survival, endoplasmic reticulum (ER) stress response gene involved in tumor cell adaptation to nutrient availability. J Biol Chem, 2014. 289(32): p. 22090-102.
88. Foufelle, F. and P. Ferre, New perspectives in the regulation of hepatic glycolytic and lipogenic genes by insulin and glucose: a role for the transcription factor sterol regulatory element binding protein-1c. Biochem J, 2002. 366(Pt 2): p. 377-91.
89. Foretz, M., et al., AMP-activated protein kinase inhibits the glucose-activated expression of fatty acid synthase gene in rat hepatocytes. J Biol Chem, 1998. 273(24): p. 14767-71.
90. Zhou, G., et al., Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest, 2001. 108(8): p. 1167-74.
91. Decaux, J.F., B. Antoine, and A. Kahn, Regulation of the expression of the L-type pyruvate kinase gene in adult rat hepatocytes in primary culture. J Biol Chem, 1989. 264(20): p. 11584-90.
Hepatic Seipin depletion increases SCD1 activity and LD formation
29
92. Prip-Buus, C., et al., Induction of fatty-acid-synthase gene expression by glucose in primary culture of rat hepatocytes. Dependency upon glucokinase activity. Eur J Biochem, 1995. 230(1): p. 309-15.
93. O'Callaghan, B.L., et al., Glucose regulation of the acetyl-CoA carboxylase promoter PI in rat hepatocytes. J Biol Chem, 2001. 276(19): p. 16033-9.
94. Koo, S.H., A.K. Dutcher, and H.C. Towle, Glucose and insulin function through two distinct transcription factors to stimulate expression of lipogenic enzyme genes in liver. J Biol Chem, 2001. 276(12): p. 9437-45.
95. Waters, K.M. and J.M. Ntambi, Insulin and dietary fructose induce stearoyl-CoA desaturase 1 gene expression of diabetic mice. J Biol Chem, 1994. 269(44): p. 27773-7.
96. Foufelle, F., et al., Glucose stimulation of lipogenic enzyme gene expression in cultured white adipose tissue. A role for glucose 6-phosphate. J Biol Chem, 1992. 267(29): p. 20543-6.
97. Jones, B.H., et al., Glucose induces expression of stearoyl-CoA desaturase in 3T3-L1 adipocytes. Biochem J, 1998. 335 ( Pt 2): p. 405-8.
Hepatic Seipin depletion increases SCD1 activity and LD formation
30
Table 1. List of antibodies used for immunoblotting
Antibody target Manufacturer Catalog number Concentration used